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Abstract

Background and Purpose—The neuroprotective potential of citicoline in acute ischemic stroke has been shown in many experimental studies and, although the exact mechanisms are still unknown, a clinical Phase III trial is currently underway. Our present study was designed to check whether citicoline also enhances neuroregeneration after experimental stroke.

Methods—Forty Wistar rats were subjected to photothrombotic stroke and treated either with daily injections of citicoline (100 mg/kg) or vehicle for 10 consecutive days starting 24 hours after ischemia induction. Sensorimotor tests were performed after an adequate training period at Days 1, 10, 21, and 28 after stroke. Then brains were removed and analyzed for infarct size, glial scar formation, neurogenesis, and ligand binding densities of excitatory and inhibitory neurotransmitter receptors.

Results—Animals treated with citicoline showed a significantly better neurological outcome at Days 10, 21, and 28 after ischemia, which could not be attributed to differences in infarct volumes or glial scar formation. However, neurogenesis in the dentate gyrus, subventricular zone, and peri-infarct area was significantly increased by citicoline. Furthermore, enhanced neurological outcome after citicoline treatment was associated with a shift toward excitation in the perilesional cortex.

Conclusions—Our present data demonstrate that, apart from the well-known neuroprotective effects in acute ischemic stroke, citicoline also possesses a substantial neuroregenerative potential. Thanks to its multimodal effects, easy applicability, and history as a well-tolerated drug, promising possibilities of neurological treatment including chronic stroke open up.

Introduction

Although most patients with stroke show some degree of spontaneous functional improvement,1 recovery is generally far from complete. Therefore, there is an urgent need to identify therapeutic strategies to support endogenous poststroke repair mechanisms. One candidate stroke drug for ischemic stroke with an extended therapeutic window, which is currently under investigation in a randomized, double-blind, placebo-controlled, multicenter clinical Phase III trial (International Citicoline Trial on Acute Stroke [ICTUS]), is citicoline (also known as cytidine-5-diphosphocholine or CDP-choline). Although citicoline seems to display a multitude of beneficial effects, the exact mechanisms of action are still enigmatic.

Citicoline, a naturally occurring endogenous compound, is an essential intermediate in the biosynthesis of phosphatidylcholine.2 Citicoline has been shown to have neuroprotective effects in a variety of central nervous system injury models, including cerebral ischemia.3–6 The suggested mechanisms that may explain the neuroprotective actions of citicoline include prevention of fatty acid release,7 stimulation of phosphatidylcholine synthesis,2 preservation of cardiolipin and sphingomyelin levels,7 increase of glutathione synthesis and glutathione reductase activity,8 restoration of Na+/K+-ATPase activity,9 and antiglutamatergic effects.10,11 In addition to its neuroprotective effects, citicoline has convincingly been shown to also have neuroregenerative effects12 although, here again, the underlying mechanisms are still largely unknown.

In the present study, in addition to the analysis of the impact of citicoline on the long-term functional outcome, we particularly explored the neuroregenerative effect of citicoline on poststroke neurogenesis in the subventricular zone (SVZ), the dentate gyrus (DG) of the hippocampus, and the peri-infarct area (PI). Because citicoline exhibits membrane-stabilizing properties,13 we further checked whether citicoline may cause alterations in the abundance of excitatory glutamate or the inhibitory γ-aminobutyric acid type A (GABAA) receptor in perilesional and/or remote areas possibly contributing to behavioral recovery.

Materials and Methods

Animals

A total of 40 male Wistar rats (Charles River, Sulzfeld, Germany), weighing 180 to 200 g on arrival, were used in the experiments. They were housed in groups of 2 animals in Macrolon cages. All rats were kept under controlled environmental conditions (ambient temperature 22°C, 12-hour light/dark cycle, lights on at 7:00 am). Standard laboratory chow (Altromin1324, Lage, Germany) and tap water were given ad libitum.

Photothrombotic Ischemia Model

All experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and experimental protocols were approved by the local ethics committee. Experiments were performed on adult male Wistar rats (270–310 g). Animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight; Ketanest) and xylazine hydrochloride (8 mg/kg body weight). The left femoral vein was cannulated with a PE-50 tube for Bengal Rose infusion. The rectal temperature was maintained at 37°C by a thermostat-controlled heating pad (Föhr Medical Instruments). Photothrombotic ischemia was induced in the right frontal cortex. For illumination, a laser spot of 8 mm in diameter (G Laser Technologies) was placed stereotaxically onto the skull 0.5 mm anterior to the bregma and 3.5 mm lateral from the midline. The skull was illuminated for 20 minutes. During the first 2 minutes of illumination, the dye Bengal Rose (0.133 mL/kg body weight, 10 mg/mL saline) was injected intravenously. Six animals died during surgeries. After being operated on, all the animals were recoded by an assistant to ensure necessary blinding.

Treatment

Animals were randomly assigned to the treatment and control groups, respectively, receiving daily injections either of citicoline (100 mg/kg; remaining: n=17) or vehicle (saline; remaining: n=17) for 10 consecutive days starting 24 hours after induction of the ischemia. To label dividing cells, each animal received a daily bromodeoxyuridine (BrdU) injection (50 mg/kg per day intraperitoneally) throughout the 10-day treatment period, 1 hour before the respective citicoline or saline injection.

Behavioral Testing

The behavioral experiments were performed on a total number of 34 animals (citicoline-treated group n=17; control group n=17). In all animals, the adhesive-tape removal test and the cylinder test were performed 1 day before ischemia (baseline) after a training period of 3 days and at Days 1, 10, 21, and 28 after ischemia by an investigator blinded to the experimental groups. For further details, see the online-only Data Supplement.

Immunohistochemistry for Analysis of Neurogenesis

For immunohistological analysis of neurogenesis, the brains of 14 animals were used (n=7 per group). Twenty-eight days after ischemia, animals were reanesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 mol/L phosphate buffer. The brains were fixed overnight in 4% paraformaldehyde at 4°C. The tissue was then cryoprotected by 3-day immersion in 30% sucrose solution and stored at −80°C until analysis. Immunohistochemistry was performed on sagittal free-floating 40-μm sections with the following antibodies: rat anti-BrdU (1:500; Abcam), mouse antineuronal nuclei (anti-NeuN; 1:200; Millipore), and rabbit antidoublecortin (1:500; Abcam). BrdU/NeuN-positive cells were analyzed in 3 brain regions: DG, SVZ, and PI. In the DG and SVZ, all BrdU-positive cells were counted on 7 sections (every 12th section, 440-μm intervals) per hemisphere. For the analysis of BrdU/NeuN-positive cells in the PI, 4 squares (300 μm×300 μm) adjacent to the PI were analyzed on 4 sections (bregma 1 mm to −0.5 mm). To determine the percentage of neurons among the newly generated cells, 50 randomly selected BrdU-positive cells within the DG, SVZ, and PI, respectively, were analyzed for BrdU/NeuN colabeling. Multiplying the total number of BrdU-positive cells with the percentage of NeuN/BrdU, double-positive cells yielded the number of new neurons in the respective areas.

After decapitation, brains were rapidly removed, frozen for 10 minutes in isopentane at −30°C, and afterward stored at −80°C until use. Brains were then serially cut on a cryostat at −20°C starting at bregma 12.72 mm into 20-μm-thick coronal sections, which were mounted on triethoxysilyl propylamine-coated slides. For analysis of morphology and infarct volume, sections at the interaural coordinates 12.72, 10.72, 8.72, 6.72, 4.72, and 2.72 were used, respectively. Immunohistochemistry as well as in vitro receptor autoradiography was performed on slices cut at the level of the largest cortical infarct volume, normally at the level of the caudatoputamen and anterior commissure as well as at the level of the dorsal hippocampus.

Brain Tissue Calculations

Hematoxylin and eosin staining was performed according to standard protocols. Sections were scanned under equal lighting conditions with the digital CoolSNAP camera (Roper Scientific, Photometrics CoolSNAP™cf, Ottobrunn/Munich, Germany) and digitized with the MCID image analysis system (Imaging Research Inc, St Catharines, Ontario, Canada); the distance between respective coronal sections was used to calculate a linear integration for the lesion volume determinations. The investigator performing the analysis was blinded to group identity.

Analysis of dendritic integrity, microglial activation, and glial scar formation was immunohistochemically performed using antibodies against microtubule-associated protein 2, ionized calcium-binding adaptor molecule 1, and glial fibrillary acidic protein, respectively. Immunostained brain sections at the infarct level were scanned at a magnification of ×2.5 with a Leica Microscope (Leica, Germany), digitized, and transferred to a computer screen. Brightness, gain, and contrast were all kept constant during image acquisition. The glial scar and the zone of activated microglia surrounding the lesion were divided into (1) the basal part between the lesion and the corpus callosum; and (2) the lateral part of the scar representing the equivalent of the immediate border zone which was analyzed for microtubule-associated protein 2 immunoreactivity (Figure 1); both regions were marked on the monitor. Regions of interest were analyzed by densitometry using the MCID image analysis system (Imaging Research Inc) as previously described.14 The investigator performing the analysis was blinded to group identity. For further details, see the online-only Data Supplement.

Receptor binding densities were analyzed at the level of the largest infarct area in the immediate border zone, border zone, adjacent motor cortex (AMC), and remote primary somatosensory cortex upper lip (S1ULp; left) as well as in corresponding contralateral regions (not shown). In addition, receptor binding densities in remote somatosensory cortex barrel field (S1Bf), somatosensory cortex trunk region (S1Tr), and secondary somatosensory cortex (S2) were analyzed at the level of the dorsal hippocampus ipsilaterally (right) and contralaterally (not shown).

In Vitro Receptor Autoradiography

Quantitative in vitro receptor autoradiography studies were performed using [3H]MK-801, [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and [3H]muscimol for labeling of N-methyl-d-aspartate (NMDA), AMPA, and GABAA receptors, respectively. Ligands were purchased from Perkin Elmer, Inc. Labeling and incubation procedures for the different binding sites were performed according to the protocols of Zilles and colleagues15 as previously described.16,17 For further details, see the online-only Data Supplement. Quantitative analysis of ligand binding was performed in the following regions of interest ((Figure 1): (1) in the lateral peri-infarct area including the “immediate border zone” (a lateral thin rim close to the lesion), in the “border zone,” and in the adjacent motor cortex (M1/M2) as well as in corresponding regions of the contralateral hemisphere, respectively; and (2) in various remote cortical regions both ipsi- and contralateral to the lesion, namely the upper lip of the primary somatosensory cortex, the trunk region, the barrel field (S1Ulp, somatosensory cortex trunk region, S1BF), and the secondary somatosensory cortex. The regions of interest were marked on the monitor and the gray values were automatically assessed by the imaging software. Nonspecific binding was just above background labeling or completely invisible; thus, background density was used as an estimate of the optical density (OD) of unspecific binding (ODUSP) and was subtracted from total binding (ODTOT), resulting in specific OD (ODSP). Ligand binding densities in citicoline- and placebo-treated rats were analyzed by calculating mean concentration values for each ligand and region. OD values are means±SD presented as percent of control rats. Final values were normalized to control levels (mean±SD) of the control animals for each experiment as described before.17 The investigator performing the analysis was blinded to group identity.

Statistical Analysis

Randomization was carried out by the computer software “Research Randomizer” (Version 3.0; Urbaniak GC, Plous S, 2011, retrieved on April 22, 2011, from www.randomizer.org/). The values presented in this study are means±SEM. Statistical analyses were calculated using the Statistical Package of Social Sciences (Version 15.0; SPSS Inc, Chicago, IL). The normality distribution of the data was assessed by graphical examination of the histograms and verified by the Shapiro-Wilk test (P>0.05). Sensorimotor measurements were analyzed by 2-way repeated-measures analysis of variance followed by the Fisher protected least significant difference test. Student t test with Bonferroni correction was used to compare data between 2 groups. An α error rate of 0.05 was taken as the criterion for significance.

The normality distribution of the data for brain tissue calculations and receptor autoradiography were verified by the Shapiro-Wilk test (P<0.05). Significant group effects were tested by analysis of variance. Post hoc analysis was performed where appropriate by pairwise t tests between the groups. A probability value <0.05 was considered statistically significant. Analysis was performed using the general statistics module of Analyze-it for Microsoft Excel (Analyze-it Software, Ltd, Leeds, UK). Values are means±SD presented as percent of control rats.

Results

Functional Outcome

The photothrombotic stroke model causes distinct deficits in somatosensory and motor functions. In the present study, somatosensory recovery was assessed by the adhesive-tape removal test and motor recovery by means of the cylinder test. The animals treated with citicoline had a more favorable somatosensory recovery compared with the control group (Figure 2A). This effect reached significance (P<0.05, Fisher protected least significant difference post hoc test after significant 2-way repeated-measures analysis of variance) on Day 10 after ischemia and persisted until the end of the experiment (Day 28). In analogy to the improved somatosensory function, the animals treated with citicoline exhibited enhanced motor recovery compared with the control group (Figure 2B). This effect reached significance (P<0.05, Fisher protected least significant difference post hoc test after significant 2-way repeated-measures analysis of variance) on Day 21 after ischemia and also persisted until the end of the experiment (Day 28).

Functional testing: (A) somatosensory recovery was assessed by the adhesive-tape removal test. Animals treated with citicoline showed enhanced somatosensory recovery compared with the control group. Note the significant improvement starting on Day 10 until Day 28 after ischemia. B, Motor recovery was assessed by the cylinder test. Note the significant improvement in motor function of the animals treated with citicoline compared to controls, starting on Day 21 until Day 28 after ischemia. (means±SEM; *P<0.05, Fisher protected least significant difference post hoc test after significant 2-way repeated-measures analysis of variance).

Detection of Postischemic Neurogenesis

Our analyses disclosed a significant increase in newborn neurons in the DG, SVZ, and PI of citicoline-treated animals compared with controls 28 days after ischemia (Figure 3). Further analyses revealed a significant increase in the amount of BrdU-positive cells in the DG (161.07±36.96 versus 67.25±16.48) and SVZ (154.57±19.83 versus 93.25±12.07) of citicoline-treated compared with vehicle-treated animals, respectively. This increase in BrdU single-positive cells could not be detected in the PI citicoline compared with the control group (411.14±18.13 versus 377.88±13.93, respectively; Bonferroni-corrected Student t test, P=0.16). To investigate possible neuron-specific effects of citicoline treatment, we analyzed the percentage of BrdU/NeuN-positive cells in the DG, SVZ, and PI. Citicoline treatment led to a significantly higher percentage of BrdU/NeuN double-positive cells in the DG (75±4.36 versus 53.75±2.63) as well as in the SVZ (70.71±6.12 versus 51.25±3.75) and in the PI (58.29±3.37 versus 36.12±3.05). Furthermore, doublecortin immunohistochemistry visualized the migration of doublecortin-positive neuronal precursor cells from the SVZ to the PI (Figure 3).

Brain Tissue Calculations

One of 10 mice of the saline-treated control group was excluded from further analysis because there was no cerebral infarct detectable. Total infarct volumes did not significantly differ between the control and citicoline-treated groups (8.15±2.38 mm3 versus 10.78±4.76 mm3, P=0.154). Volumes of the total contralateral as well as the total ipsilateral hemispheres did not significantly differ between the control and citicoline-treated groups (contralateral: 341.80±20 mm3 versus 350.57±11.62 mm3, P=0.258; ipsilateral: 329.47±18.42 mm3 versus 333.69±10.95 mm3, P=0.547). Moreover, analysis of the remaining ipsilateral brain tissue revealed nonsignificant differences between controls and citicoline-treated animals (321.31±16.92 mm3 versus 322.91±10.37 mm3; P=0.804).

Dendritic Integrity in the Peri-Infarct Region

Microtubule-associated protein 2-stained sections for assessment of dendritic integrity did not reveal significant differences between controls (set as 100%) and citicoline-treated rats both in the immediate border zone (100.0%±11.1% versus 115.4%±22.6%, P=0.082) and in the border zone (100.0%±12.9% versus 95.1%±11.7%, P=0.397).

Scar Formation in the Peri-Infarct Area

Glial fibrillary acidic protein-stained sections for assessment of glial scar formation did not reveal significant differences between controls (set as 100%) and citicoline-treated rats both in the basal (100.0%±27.4% versus 114.3%±36.7%, P=0.353) and lateral part (100.0%±29.7% versus 111.5%±41.1%, P=0.909).

In Vitro Receptor Autoradiography

Ligand Binding of [3H]MK-801, [3H]AMPA, and [3H]Muscimol to NMDA, AMPA, and GABAA Receptors, Respectively, in the Peri-Infarct Region

[3H]MK-801 receptor binding was significantly increased in the immediate border zone in citicoline-treated animals compared with saline-treated animals (P=0.0338). In the border zone of citicoline-treated rats, [3H]MK-801 binding was also slightly increased compared to saline-treated controls, but these difference did not reach significance. Similarly, [3H]AMPA receptor binding was also significantly increased in the immediate border zone only in citicoline-treated animals (P=0.0448), whereas the slight increase of [3H]AMPA receptor binding in the border zone did, again, not reach a significant level. However, [3H]AMPA binding values in citicoline-treated animals were significantly reduced in the contralateral cortex corresponding to the region of the border zone compared with controls (P=0.0321). [3H]muscimol receptor binding to inhibitory GABAA receptors did not significantly differ in the peri-infarct regions and the corresponding contralateral cortical areas comparing both treatment groups (Figure 4).

[3H]MK-801, [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and [3H]muscimol ligand binding to N-methyl-d-aspartate (NMDA), AMPA, and γ-aminobutyric acid type A (GABAA) receptors, respectively, in the perilesional immediate border zone and border zone of citicoline-treated rats compared with controls. Values in percent of control level are presented on the left. Representative color-coded autoradiographs of [3H]MK-801, [3H]AMPA, and [3H]muscimol ligand-binding at the level of the largest infarct volume after citicoline treatment compared with controls are presented on the right side. The scale on the right indicates the relative ligand binding densities from low (purple) to high (red; means±SD; *P<0.05, analysis of variance and Bonferroni error protection).

Ligand Binding of [3H]MK-801, [3H]AMPA, and [3H]Muscimol to NMDA, AMPA, and GABAA Receptors, Respectively, in the Adjacent Motor Cortex

Although [3H]MK-801 ligand binding densities in the motor cortex adjacent to the lesion were not significantly altered by citicoline treatment, [3H]AMPA ligand binding was reduced throughout all layers both ipsi- and contralateral to this lesion but reached significance only in contralateral Layer IV (P=0.0167; Figures 5 and 6). In addition, citicoline treatment induced a slight overall increase of [3H]muscimol ligand binding densities in the motor cortex but only the increased binding in ipsilateral Layer I was significant compared with controls (P=0.0171).

Primary Somatosensory Cortex Upper Lip

Ipsilateral cortical [3H]MK-801 receptor binding values did not significantly differ between the 2 experimental groups. Apart from a significant decrease in contralateral primary somatosensory cortex upper lip Layer III (P=0.0042) in the citicoline-treated group, no significant differences were detectable in the contralateral hemisphere. [3H]AMPA receptor binding was significantly increased in ipsilateral primary somatosensory cortex upper lip Layer I (*P=0.0151) in citicoline-treated rats compared with controls, whereas [3H]AMPA binding values in all the other layers both ipsi- and contralateral did not significantly differ between the citicoline-treated and control groups. [3H]muscimol binding was largely identical in both groups throughout all the layers in both hemispheres (Figures 5 and 6).

Primary Somatosensory Cortex Trunk Region

In the animals treated with citicoline, [3H]MK-801 binding densities were significantly reduced only in contralateral somatosensory cortex trunk region Layer IV (P=0.0386), whereas the other contralateral layers as well as all ipsilateral layers showed no significant differences compared with controls (P>0.05). [3H]AMPA binding densities were slightly reduced in both hemispheres of citicoline-treated rats and reached significance in contralateral Layers II (P=0.0248) and IV (P=0.0345) compared with the saline-treated controls. In both experimental groups, [3H]muscimol ligand binding densities did not differ significantly (Figures 5 and 6).

Secondary Somatosensory Cortex

[3H]MK801 ligand binding in Layers I to VI of both hemispheres was not significantly altered by citicoline treatment compared with controls, whereas [3H]AMPA ligand binding was slightly reduced throughout all layers both ipsi- and contralateral after citicoline treatment, although these differences reached significance only in the ipsilateral secondary somatosensory cortex Layer VI (P=0.0054). The experimental groups did not significantly differ in [3H]muscimol ligand binding in all layers of the secondary somatosensory cortex region of both hemispheres (Figures 5 and 6).

Discussion

Focusing on the regenerative potential of delayed citicoline treatment in a photothrombotic stroke model, we could demonstrate a robust improvement of sensorimotor recovery 21 and 28 days after ischemia, respectively. As 1 mechanism contributing to this more favorable neurological outcome, we could identify increased neurogenesis in the SVZ and migration of neural progenitors to the lesion with increased neurogenesis also within the PI. A second component of the regeneration-enhancing effect of citicoline was a shift toward excitation in the perilesional cortex.

In our present study, treatment with citicoline resulted in a significantly improved functional recovery compared with placebo measured by both the adhesive-tape removal test and the cylinder test, which reliably disclose even slight sensorimotor deficits in the photothrombotic stroke model.18 Pharmacological stimulation with citicoline alleviated postischemic functional deficits, although treatment was initiated not until 24 hours after onset of ischemia, that is, beyond established time points of neuroprotection but corresponding to the time window in the current clinical citicoline multicenter study (ICTUS). Importantly, these beneficial citicoline effects were neither attributable to differences in cortical lesion volumes and the remaining brain tissue nor to differences in perilesional astroglial scar formation. Only the microglial response surrounding the lesion was reduced in the citicoline group. Because microglia is thought to mediate beneficial effects beyond acute ischemic stroke, this finding suggests that other, more powerful mechanisms are also induced by citicoline.19

Neurogenesis is suggested to play a decisive role in mediating functional recovery after experimental stroke.20 Within the healthy brain, adult neurogenesis supplies newborn neurons from the subgranular zone to the adjacent dentate gyrus of the hippocampus and from the SVZ to the olfactory bulb (for an in-depth review, see Zhao et al21). Stroke alters this normal pattern of adult neurogenesis to stimulate cell proliferation within the SVZ and subgranular zone and migration of newborn, immature neurons from the SVZ into areas of injury.22,23 Although a recent study by Osman and colleagues24 compellingly demonstrated that the injured cortex continuously receives new progenitors from the SVZ up to 1 year after photothrombotic stroke, we, here, have demonstrated, for the first time, that citicoline has a major impact on postischemic neurogenesis and the migratory response to the ischemic lesion. Citicoline treatment caused an increase in BrdU/NeuN-positive cells in the DG of the hippocampal formation, in the SVZ, and in the PI. In analogy to the findings originating from Osman and coworkers, we could show migration of doublecortin-positive neuronal precursor cells from the SVZ to the lesion 28 days after photothrombotic stroke. At first glance, the neuroregenerative effects of newborn neurons seem to be limited because the majority of these newborn neurons undergo apoptosis at a progenitor and young neuronal stage.25,26 However, treatment with trophic factors such as the brain-derived neurotrophic27–28 and the granulocyte colony-stimulating factor29 supports the survival of newborn neurons, thereby promoting the neuroregenerative effect of neurogenesis. Apart from purely correlative studies, there is strong evidence of a causal connection between neurogenesis and the increase in poststroke recovery. Ablation of neural precursors, expressing doublecortin before permanent middle cerebral artery occlusion in a doublecortin–thymidine kinase transgenic mouse model, resulted in exaggerated postischemic sensorimotor deficits.30 Therefore, attenuation of sensorimotor defects by citicoline, in association with enhanced neurogenesis, strongly argues for the regenerative potential of this substance. These results extend the recent findings from Hurtado and colleagues12 who could demonstrate that chronic postischemic treatment with citicoline improves functional recovery associated with modification of pre-existing neuronal structures such as an increase in dendritic complexity and spine density of pyramidal neurons of Layer V in the contralateral sensorimotor cortex.

Because citicoline has distinctive membrane-modulating properties,13 the abundance of molecules such as membrane-bound receptors may change after application of this drug. Therefore, we checked for alterations of excitatory and inhibitory neurotransmitter receptor ligand-binding densities as 1 potential component of the plastic response induced by citicoline. There is substantial evidence that the neuroprotective effect of citicoline in acute stroke models is partially due to its antiglutamatergic effects.10,11 Even in vitro, citicoline was able to protect motor neurons31 as well as cerebellar granule cells32 against glutamate-mediated apoptosis. Furthermore, administration of the NMDA receptor antagonist MK-801 together with citicoline showed synergistic neuroprotective effects 7 days after temporary middle cerebral artery occlusion.33 However, over the past years, it has become clear that the excitotoxic postischemic phase, characterized by overactivation of glutamate receptors, is surprisingly short. Using a mouse model of head injury, Biegon and colleagues34 could convincingly demonstrate that hyperactivation of NMDA receptors occurred only during the first hour after the excitotoxic stimulus but then was followed by a profound and long-lasting functional loss. Consequently, stimulation of NMDA receptors 24 and 48 hours after injury significantly improved functional outcome. Similarly, transient focal cerebral ischemia also reduced [3H]MK801 ligand binding densities35 and, again, treatment with an NMDA agonist did enhance the neurological outcome.36 Our findings in the citicoline-treated group with increased perilesional NMDA and AMPA receptor-binding densities 28 days after photothrombotic stroke, exhibiting improved neurological outcome, fit well to the hypothesis that citicoline may be a safe drug not interfering with ongoing regenerative processes. These data are also in accordance with previous studies from our laboratories where, in the same ischemia model, after treatment with 2 different growths factors, brain-derived neurotrophic factor and granulocyte-colony stimulating factor, the best poststroke performance was also associated with an increase in or, at least, maintenance of perilesional ligand-binding densities of excitatory glutamate receptors.18 These findings further corroborate the hypothesis that, during the chronic phase after stroke, postischemic hyperexcitability may enhance functional outcome in the long run.37

Summary

In conclusion, our present data demonstrate that, apart from its well-known neuroprotective effects in acute ischemic stroke, citicoline also possesses a substantial neuroregenerative potential. Effectiveness even after delayed application 24 hours after onset of ischemia opens up promising possibilities also in the treatment of chronic stroke.

Sources of Funding

This study was supported by Trommsdorff GmbH & Co KG Arzneimittel.

Disclosures

Acknowledgments

The present study includes parts of the doctoral thesis of B.K.S. The technical expertise of Nicole Roder, Maike Hoppen, and Birgit Geng is kindly acknowledged. Furthermore, we thank Astrid Wöber for her editorial assistance.

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